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The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins
Biochimie 84 (2002) 391–397
The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins Jean-Claude Lazzaroni *, Jean-François Dubuisson, Anne Vianney Unité de Microbiologie et Génétique, UMR5122 CNRS-INSA, Université Lyon-1, bâtiment André-Lwoff, 10, rue Dubois, 69622 Villeurbanne cedex, France Received 25 February 2002; accepted 7 June 2002
Abstract The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Tol–Pal proteins; Group A colicins; Energy transduction; Signal transduction
1. Introduction Colicins are bacterial protein toxins, which are active against Escherichia coli and other related species. They use different ways to kill susceptible cells, ranging from the ability to depolarize the cytoplasmic membrane, to cytotoxic activity against cytoplasmic nucleic acids, or interference with cytoplasmic membrane transporters like bactoprenyl phosphate [1–4] (Table 1). The mechanisms by which these folded proteins are able to cross the membrane barriers of the target cells have been extensively studied. Colicins first recognize an outer membrane receptor, then they interact with proteins in the periplasmic space to reach their target. Group A colicins use the Tol system for their translocation, while group B colicins require the Ton system. Among group A colicins, the most studied are E colicins which bind the BtuB outer membrane protein, the high affinity transporter of vitamin B12. Colicins are made of a central receptor-recognition domain that binds the outer membrane receptor and an N-terminal translocation domain that interacts with periplasmic proteins triggering the movement of the C-terminal cytotoxic domain into the cell [3,4].
* Corresponding author. Tel.: +33-472-43-1367; fax: +33-472-43-2686. E-mail address: [email protected] (J.C. Lazzaroni).
The only published structures of full-length colicins are those of colicin Ia [5] and E3 [6]. They are in agreement with this domain organization. They present a hairpin like structure with each of the three functional domains separated by long α-helices able to span the periplasm. This review focuses on the role of the Tol proteins in the translocation of group A colicins. The Tol system comprises five proteins, TolQ, TolR, TolA, TolB and Pal. The tolQRAB and pal genes are clustered in the genomes of most Gram-negative bacteria so far sequenced [7]. In E. coli, they are transcribed from two promoters, giving the ybgC–tolQ–tolR–tolA–tolB–pal–ybgF and tolB–pal–ybgF transcripts [8,9]. No obvious phenotype has been assigned to ybgC and ybgF which encode a cytoplasmic and a periplasmic protein, respectively. Mutations in any of the tol–pal genes result in hypersensitivity to deleterious agents, release of periplasmic content, formation of outer membrane vesicles at the cell surface and induction of capsule synthesis which results in a mucoid phenotype [10]. The translocation of filamentous phage DNA and group A colicins requires the TolQRA proteins [11–13]. Some group A colicins also need TolB for this purpose (Table 1). The Ton system consists of three proteins, TonB, ExbB and ExbD [14]. The corresponding genes are not always clustered in the genomes of Gram-negative bacteria. Mutations in these genes affect the active transport of iron
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 1 9 - 0
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Table 1 Group A colicins use different receptors and translocation proteins to penetrate bacteria Colicin
Receptor
Translocation
Cytotoxic activity
A E1 E2, E7, E8, E9 E3, E4, E6 E5 N K U, 28b DF13 .
BtuB BtuB BtuB BtuB BtuB OmpF Tsx OmpA IutA
OmpF, TolQRAB TolC, TolQRA OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB TolQRA OmpF, OmpA, TolQRAB OmpF LPS TolQRAB TolQRA
Pore forming Pore forming Dnase 16s rRNase anticodon rRNase Pore forming Pore forming Pore forming 16s rRNase
siderophore and vitamin B12. They are required for the entry of group B colicins and of T1 and Φ80 phage DNA. TolQR and ExbBD share homologies while only a SHLS motif is conserved in the N-terminal domains of TolA and TonB [15–17].
2. The Tol proteins TolQ, TolR and TolA are cytoplasmic membrane proteins (Fig. 1). TolQ is an integral inner membrane protein containing three transmembrane domains with two cytoplasmic regions, one between helices 1 and 2 and the other at the C-terminus [18]. TolR and TolA are anchored to the cytoplasmic membrane by a single membrane spanning segment near the N-terminus, leaving most of the protein exposed to the periplasm [19,20]. TolR and TolA have a three-domain structure [19,21]. In addition to the N-terminal anchoring region, TolA usually contains a large central domain with a high degree of α-helical structure and a C-terminal domain whose crystal structure has been established [22,23]. Each of the three domains is separated by a stretch of glycine residues, which confer some flexibility to the protein. TolR also has an N-terminal anchoring domain, a central domain and a C-terminal domain which has been proposed to form an amphiphatic helix interacting with the cytoplasmic membrane [21]. TolQ, TolR and TolA form a complex in the cytoplasmic membrane (Fig. 1). Biochemical and genetic studies have shown that these interactions involve the transmembrane domains of the three proteins along with the C-terminal amphiphatic helix of TolR [24,25]. The crystal structure of TolB, a periplasmic protein, has been determined [26,27]. It contains an N-terminal α + β domain based on a five-stranded mixed β-sheet and a C-terminal six-bladed β-propeller domain. Pal is an outer membrane peptidoglycan-associated lipoprotein [28]. It is anchored to the outer membrane by its N-terminal lipid moiety and strongly interacts with the peptidoglycan layer by its carboxy-terminal region which contains a particular sequence motif [29]. Pal also forms a complex with TolB [30]. The same region of Pal interacts with the β-propeller domain of TolB and the peptidoglycan [31,32]. These
interactions appear to be mutually exclusive since a TolB–Pal complex is not associated with the peptidoglycan [33]. Cell fractionation experiments suggest that the Tol–Pal proteins are preferentially associated with contact regions between the inner and outer membrane [34]. E. coli contains approximately 1000 group A colicin import sites [35]. The stoichiometry of each of the Tol–Pal proteins is not well defined. Quantification studies suggest that Pal is in excess over the TolA and TolR inner membrane components [19,20,36,37]. This is in agreement with the genetic orga nization of the tol–pal region which contains an internal promoter upstream tolB.
Fig. 1. Localization of the Tol–Pal proteins in the cell envelope of Gram-negative bacteria.
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Recently, energy-dependent conformational changes in TolA have been characterized. They depend on the transmembrane domain of TolA and of TolQ and TolR proteins [38]. The transmembrane fragment of TolR and the third transmembrane fragment of TolQ are involved in the pmf-dependent activation of TolA [39]. TolQ and TolR present structural and functional homologies not only with ExbB and ExbD but also with MotA and MotB, components of the flagellar motor, raising the attractive hypothesis that the transmembrane domains of TolQ, TolR and TolA constitute an ion potential-driven molecular motor [40]. The TolQRA and TolBPal complexes are connected by the interaction between Pal and TolA which requires the proton motive force, TolQ and TolR proteins [39]. In addition, the C-terminal domain of TolA interacts with the N-terminal domain of TolB [41,42]. Interestingly, in the case of the flagellar motor, MotB not only shares homologies with TolR and ExbB, but also contains the particular peptidoglycanassociating motif common with Pal and OmpA at its C-terminus [29]. Thus, this protein may contribute to link the peptidoglycan to the inner membrane in a way similar of that used by the Tol–Pal proteins to link the outer membrane to the peptidoglycan. All these results are consistent with the fact that TolA activation which requires a functional Tol cytoplasmic membrane complex, drives a signal to Pal via a change of conformation of its C-terminal domain, generating a transient interaction between the Tol cytoplasmic and outer membrane complexes. Interactions between Pal, TolB and other peptidoglycanassociated proteins have also been demonstrated [31,36]. The three most abundant outer membrane proteins interacting with the peptidoglycan network, namely Lpp, OmpA and Pal, together with TolB interact and might constitute a structural network to link the peptidoglycan and the outer membrane. Therefore, the physiological role of the Tol–Pal system could be to participate to this network via the TolBPal complex.
3. The translocation of group A colicins It is considered that very few molecules of colicin are sufficient to kill target cells. To investigate the interaction between colicins and proteins involved in their translocation, two methods have been used, overexpression of the N-terminal translocation domain of colicins into the periplasm [43,44], or purification of the same domain for in vitro biophysical studies [45]. Before their translocation, colicins bind to their receptor. Unfolding of colicin A after binding to its receptor has been demonstrated [46]. Most colicins use an outer membrane porin as receptor (Table 1). In the case of E colicins which use BtuB, a 76-residue polypeptide of the central domain of colicin E9 confers receptor specificity and competes with the transport of vitamin B12, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive [47]. One
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possibility is that the N-terminal domain of colicins crosses the membrane near or through these proteins to contact a Tol protein. In the case of some group B colicins which use the large channel-forming receptors FhuA and FepA, the possibility that the polypeptide chain crosses the outer membrane through the channel has been suggested [48]. The information for the translocation step of colicins is included in their N-terminal domain [49]. It has been shown that the C-terminal domain of TolA interacts with the N-terminal domains of colicins A, and N [45,50]. The same region of TolA is involved in the interaction with protein g3p of the filamentous phage f1 [23]. Some colicins also interact with the C-terminal domain of TolB [27,43,51] and the central domain of TolR [51]. In the case of colicin A, the region of interaction with TolB, called the “TolB box”, is between residues 7 and 20 [43,51], the region of interaction with TolA consists of residues 52 and 97 [43], while residues 7–14 are involved in the interaction with TolR [51]. The TolB-box is conserved in several colicins that need TolB for their translocation step [27,43,44]. There is no sequence homology in the regions of interaction with TolA except for colicins A and K which suggests that these colicins interact with TolA in a similar way [52]. In the case of colicin N, the minimal TolA binding region is 27 residues [53]. Colicin E1 interacts with TolA differently than the other group A colicins [54]. The regions of interaction between TolB and TolR clearly overlap, suggesting that interaction between colicins and TolB and TolR is not simultaneous. This is not the case for the interactions with TolA and TolB since a ternary complex between colicin A, TolB and TolA has been characterized [43]. All these results have been summarized to propose a model for the translocation of colicin A (Fig. 2). The model takes into account the envelope localization of the Tol proteins and the structural information assuming that the structure of colicin A and Ia are similar. Colicin first binds to BtuB receptor via its central domain and unfolds (Fig. 2, step 1). The N-terminal translocation domain crosses the outer membrane near or in OmpF and the TolB box interacts with TolB which is close to the outer membrane due to its interaction with Pal (Fig. 2, step 2). As a consequence, TolB is no longer able to interact with TolA and Pal. This induces a tol phenotype and results in outer membrane fragility, allowing the colicin to further penetrate into the periplasm where it interacts with the C-terminus of TolA (Fig. 2, step 3). At this level a ternary complex between the colicin, TolA and TolB may occur. Colicin A then interacts with TolR near the inner membrane (Fig. 2, step 4). The mechanism of entry of the C-terminal catalytic domain is largely unknown. It is only known that colicin A spans the envelope and is still in contact with its outer membrane receptor when its translocation is completed [55]. Colicin A then forms a pore that depolarizes the inner membrane. Although this model can be generalized to colicins that use TolB for their
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Fig. 2. A proposed model for the translocation of colicin A across the cell envelope. For each step, proteins proposed to be involved in the translocation process are coloured in green. Colicin domains: T, translocation, R, receptor, C, catalytic. See text for comments of the model.
translocation, the way the colicins interact with Tol proteins may differ [4,54]. The energetics of the translocation is not fully understood. It has been reported that translocation of colicin A may be voltage-independent [56]. These experiments should probably be reconsidered in the light of the new results described above. It has been shown that the TolA C-terminal domain changes conformation when interacting with colicin A [57]. One can assume that the interaction of colicin A with the TolA C-terminal domain prevents this region of TolA from interacting with Pal and TolB, leading to the inactiva-
tion of the Tol system. Therefore, a possibility to reconcile these apparent inconsistencies would be that only the first step of colicin entry requires an energized Tol–Pal complex, a step that was probably by-passed in the experimental conditions described in Bourdineaud et al. [56]. Another interesting possibility is that presented in Journet et al. [51]. These authors propose that colicin translocation occurs by Brownian ratcheting. The forward movement of colicin would occur by simple diffusion, the Tol proteins would prevent it from moving backwards and would retain it by multiple interactions in the periplasm. This hypothesis can
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probably apply for the N-terminal domain of colicins and explain that the translocation may not require energy. However, it does not explain how the C-terminal region of colicins crosses the outer membrane. Translocation of large proteins such as colicins across bacterial envelopes is a complex mechanism. Although significant work has been done to understand this mechanism, many aspects are still unsolved. For instance, some experimental reports do not fully agree with the three functional domain organization of colicins. The central domain of colicins may not be the only region involved in receptor binding. In the case of colicin N, the C-terminal pore-forming region also binds trimeric porins [58]. A hybrid construct containing the translocation and receptor domains of the g3p protein from phage f1 and the catalytic site of colicin E3 still requires TolB for its translocation, suggesting that TolB may be also involved in the entry of the C-terminal domain of colicin E3 [59]. At least four important questions remain to be answered. (i) How does colicin cross the outer membrane after interaction with their receptor? (ii) What is the exact stoichiometry of the Tol proteins? A model has been proposed where four TolQ and two TolR proteins interact with one TolA molecule [40]. Pal and TolB may dimerize [36,37]. (iii) Is the proposed Brownian ratchet sufficient to achieve colicin translocation [51]? (iv) How do enzymatic colicins, which need to cross the inner membrane, reach their final target [60,61]? The Tol system constitutes a fascinating model to understand the mechanisms used by large molecules to cross the cell envelope. As in the case of the Ton system, the Tol system appears to use an ion potential from the inner membrane to induce a conformational change of TolA in the periplasm, leading to an interaction between inner and outer membrane proteins. This kind of signalization may contribute to the role of the Tol system in maintaining the outer membrane stability. In the case of the Ton system, an energy-dependent process involving the C-terminal domain of TonB and the N-terminal domain of outer membrane receptors, leads to the opening of closed channel, allowing the uptake of iron siderophore or cobalamin. In the case of the Tol system, it is reasonable to assume that the same kind of energy-dependent process involves the C-terminal domain of TolA and an interaction with Pal and TolB (although the energy-dependence of the interaction with TolB has not been demonstrated). The tol phenotype strongly suggests that the Tol–Pal proteins are involved in outer membrane stabilization, either by contributing to a structural network between the outer membrane and the peptidoglycan, or by contributing to the localization of some outer membrane components. These two possibilities are not mutually exclusive. Colicins use the Ton and Tol systems for their translocation into the bacterial cell. While the Ton system is clearly involved in bacterial iron uptake, the exact role of the Tol system remains to be discovered. The high degree of conservation of the tol–pal operon, and the fact that some of
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the tol–pal genes appear to be required for cell viability in organisms such as Pseudomonas aeruginosa [62], Haemophilus. ducreyi [63] or E. coli O157 [64], probably reflects an important role of the Tol system in cell envelope biogenesis. In addition, the involvement of the Tol proteins in the uptake of phage DNA is not necessarily deleterious for the bacteria, since it is required for the horizontal gene transfer of genes involved in virulence [12]. Finally, a major difference between the Ton and Tol system is that the later is likely to allow both the uptake and export of biomolecules.
Acknowledgements Work in our laboratory was supported by the Life Science Department of the CNRS, the University of Lyon and a joint BQR between the University and the INSA of Lyon. JFD has an MENRT fellowship.
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J.C. Lazzaroni et al. / Biochimie 84 (2002) 391–397 [48] X. Jiang, M.A. Payne, Z. Cao, S.B. Foster, J.B. Feix, S.M. Newton, P.E. Klebba, Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria, Science 276 (1997) 1261–1264. [49] H. Benedetti, M. Frenette, D. Baty, M. Knibiehler, F. Pattus, C. Lazdunski, Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement, J. Mol. Biol. 217 (1991) 429–439. [50] H. Benedetti, C. Lazdunski, R. Lloubes, Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system, EMBO J. 10 (1991) 1989–1995. [51] L. Journet, E. Bouveret, A. Rigal, R. Lloubes, C. Lazdunski, H. Benedetti, Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm, Mol. Microbiol. 42 (2001) 331–344. [52] H. Pilsl, V. Braun, Strong function-related homology between the pore-forming colicins K and 5, J. Bacteriol. 177 (1995) 6973–6977. [53] I. Gokce, E.M. Raggett, Q. Hong, R. Virden, A. Cooper, J.H. Lakey, The TolA-recognition site of colicin N. ITC, SPR and stopped-flow fluorescence define a crucial 27-residue segment, J. Mol. Biol. 304 (2000) 621–632. [54] S.L. Schendel, E.M. Click, R.E. Webster, W.A. Cramer, The TolA protein interacts with colicin E1 differently than with other group A colicins, J. Bacteriol. 179 (1997) 3683–3690. [55] H. Benedetti, R. Lloubes, C. Lazdunski, L. Letellier, Colicin A unfolds during its translocation in Escherichia coli cells and spans the whole cell envelope when its pore has formed, EMBO J. 11 (1992) 441–447. [56] J.P. Bourdineaud, P. Boulanger, C. Lazdunski, L. Letellier, In vivo properties of colicin A: channel activity is voltage dependent but translocation may be voltage independent, Proc. Natl. Acad. Sci. USA 87 (1990) 1037–1041.
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The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins Jean-Claude Lazzaroni *, Jean-François Dubuisson, Anne Vianney Unité de Microbiologie et Génétique, UMR5122 CNRS-INSA, Université Lyon-1, bâtiment André-Lwoff, 10, rue Dubois, 69622 Villeurbanne cedex, France Received 25 February 2002; accepted 7 June 2002
Abstract The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Tol–Pal proteins; Group A colicins; Energy transduction; Signal transduction
1. Introduction Colicins are bacterial protein toxins, which are active against Escherichia coli and other related species. They use different ways to kill susceptible cells, ranging from the ability to depolarize the cytoplasmic membrane, to cytotoxic activity against cytoplasmic nucleic acids, or interference with cytoplasmic membrane transporters like bactoprenyl phosphate [1–4] (Table 1). The mechanisms by which these folded proteins are able to cross the membrane barriers of the target cells have been extensively studied. Colicins first recognize an outer membrane receptor, then they interact with proteins in the periplasmic space to reach their target. Group A colicins use the Tol system for their translocation, while group B colicins require the Ton system. Among group A colicins, the most studied are E colicins which bind the BtuB outer membrane protein, the high affinity transporter of vitamin B12. Colicins are made of a central receptor-recognition domain that binds the outer membrane receptor and an N-terminal translocation domain that interacts with periplasmic proteins triggering the movement of the C-terminal cytotoxic domain into the cell [3,4].
* Corresponding author. Tel.: +33-472-43-1367; fax: +33-472-43-2686. E-mail address: [email protected] (J.C. Lazzaroni).
The only published structures of full-length colicins are those of colicin Ia [5] and E3 [6]. They are in agreement with this domain organization. They present a hairpin like structure with each of the three functional domains separated by long α-helices able to span the periplasm. This review focuses on the role of the Tol proteins in the translocation of group A colicins. The Tol system comprises five proteins, TolQ, TolR, TolA, TolB and Pal. The tolQRAB and pal genes are clustered in the genomes of most Gram-negative bacteria so far sequenced [7]. In E. coli, they are transcribed from two promoters, giving the ybgC–tolQ–tolR–tolA–tolB–pal–ybgF and tolB–pal–ybgF transcripts [8,9]. No obvious phenotype has been assigned to ybgC and ybgF which encode a cytoplasmic and a periplasmic protein, respectively. Mutations in any of the tol–pal genes result in hypersensitivity to deleterious agents, release of periplasmic content, formation of outer membrane vesicles at the cell surface and induction of capsule synthesis which results in a mucoid phenotype [10]. The translocation of filamentous phage DNA and group A colicins requires the TolQRA proteins [11–13]. Some group A colicins also need TolB for this purpose (Table 1). The Ton system consists of three proteins, TonB, ExbB and ExbD [14]. The corresponding genes are not always clustered in the genomes of Gram-negative bacteria. Mutations in these genes affect the active transport of iron
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 1 9 - 0
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Table 1 Group A colicins use different receptors and translocation proteins to penetrate bacteria Colicin
Receptor
Translocation
Cytotoxic activity
A E1 E2, E7, E8, E9 E3, E4, E6 E5 N K U, 28b DF13 .
BtuB BtuB BtuB BtuB BtuB OmpF Tsx OmpA IutA
OmpF, TolQRAB TolC, TolQRA OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB TolQRA OmpF, OmpA, TolQRAB OmpF LPS TolQRAB TolQRA
Pore forming Pore forming Dnase 16s rRNase anticodon rRNase Pore forming Pore forming Pore forming 16s rRNase
siderophore and vitamin B12. They are required for the entry of group B colicins and of T1 and Φ80 phage DNA. TolQR and ExbBD share homologies while only a SHLS motif is conserved in the N-terminal domains of TolA and TonB [15–17].
2. The Tol proteins TolQ, TolR and TolA are cytoplasmic membrane proteins (Fig. 1). TolQ is an integral inner membrane protein containing three transmembrane domains with two cytoplasmic regions, one between helices 1 and 2 and the other at the C-terminus [18]. TolR and TolA are anchored to the cytoplasmic membrane by a single membrane spanning segment near the N-terminus, leaving most of the protein exposed to the periplasm [19,20]. TolR and TolA have a three-domain structure [19,21]. In addition to the N-terminal anchoring region, TolA usually contains a large central domain with a high degree of α-helical structure and a C-terminal domain whose crystal structure has been established [22,23]. Each of the three domains is separated by a stretch of glycine residues, which confer some flexibility to the protein. TolR also has an N-terminal anchoring domain, a central domain and a C-terminal domain which has been proposed to form an amphiphatic helix interacting with the cytoplasmic membrane [21]. TolQ, TolR and TolA form a complex in the cytoplasmic membrane (Fig. 1). Biochemical and genetic studies have shown that these interactions involve the transmembrane domains of the three proteins along with the C-terminal amphiphatic helix of TolR [24,25]. The crystal structure of TolB, a periplasmic protein, has been determined [26,27]. It contains an N-terminal α + β domain based on a five-stranded mixed β-sheet and a C-terminal six-bladed β-propeller domain. Pal is an outer membrane peptidoglycan-associated lipoprotein [28]. It is anchored to the outer membrane by its N-terminal lipid moiety and strongly interacts with the peptidoglycan layer by its carboxy-terminal region which contains a particular sequence motif [29]. Pal also forms a complex with TolB [30]. The same region of Pal interacts with the β-propeller domain of TolB and the peptidoglycan [31,32]. These
interactions appear to be mutually exclusive since a TolB–Pal complex is not associated with the peptidoglycan [33]. Cell fractionation experiments suggest that the Tol–Pal proteins are preferentially associated with contact regions between the inner and outer membrane [34]. E. coli contains approximately 1000 group A colicin import sites [35]. The stoichiometry of each of the Tol–Pal proteins is not well defined. Quantification studies suggest that Pal is in excess over the TolA and TolR inner membrane components [19,20,36,37]. This is in agreement with the genetic orga nization of the tol–pal region which contains an internal promoter upstream tolB.
Fig. 1. Localization of the Tol–Pal proteins in the cell envelope of Gram-negative bacteria.
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Recently, energy-dependent conformational changes in TolA have been characterized. They depend on the transmembrane domain of TolA and of TolQ and TolR proteins [38]. The transmembrane fragment of TolR and the third transmembrane fragment of TolQ are involved in the pmf-dependent activation of TolA [39]. TolQ and TolR present structural and functional homologies not only with ExbB and ExbD but also with MotA and MotB, components of the flagellar motor, raising the attractive hypothesis that the transmembrane domains of TolQ, TolR and TolA constitute an ion potential-driven molecular motor [40]. The TolQRA and TolBPal complexes are connected by the interaction between Pal and TolA which requires the proton motive force, TolQ and TolR proteins [39]. In addition, the C-terminal domain of TolA interacts with the N-terminal domain of TolB [41,42]. Interestingly, in the case of the flagellar motor, MotB not only shares homologies with TolR and ExbB, but also contains the particular peptidoglycanassociating motif common with Pal and OmpA at its C-terminus [29]. Thus, this protein may contribute to link the peptidoglycan to the inner membrane in a way similar of that used by the Tol–Pal proteins to link the outer membrane to the peptidoglycan. All these results are consistent with the fact that TolA activation which requires a functional Tol cytoplasmic membrane complex, drives a signal to Pal via a change of conformation of its C-terminal domain, generating a transient interaction between the Tol cytoplasmic and outer membrane complexes. Interactions between Pal, TolB and other peptidoglycanassociated proteins have also been demonstrated [31,36]. The three most abundant outer membrane proteins interacting with the peptidoglycan network, namely Lpp, OmpA and Pal, together with TolB interact and might constitute a structural network to link the peptidoglycan and the outer membrane. Therefore, the physiological role of the Tol–Pal system could be to participate to this network via the TolBPal complex.
3. The translocation of group A colicins It is considered that very few molecules of colicin are sufficient to kill target cells. To investigate the interaction between colicins and proteins involved in their translocation, two methods have been used, overexpression of the N-terminal translocation domain of colicins into the periplasm [43,44], or purification of the same domain for in vitro biophysical studies [45]. Before their translocation, colicins bind to their receptor. Unfolding of colicin A after binding to its receptor has been demonstrated [46]. Most colicins use an outer membrane porin as receptor (Table 1). In the case of E colicins which use BtuB, a 76-residue polypeptide of the central domain of colicin E9 confers receptor specificity and competes with the transport of vitamin B12, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive [47]. One
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possibility is that the N-terminal domain of colicins crosses the membrane near or through these proteins to contact a Tol protein. In the case of some group B colicins which use the large channel-forming receptors FhuA and FepA, the possibility that the polypeptide chain crosses the outer membrane through the channel has been suggested [48]. The information for the translocation step of colicins is included in their N-terminal domain [49]. It has been shown that the C-terminal domain of TolA interacts with the N-terminal domains of colicins A, and N [45,50]. The same region of TolA is involved in the interaction with protein g3p of the filamentous phage f1 [23]. Some colicins also interact with the C-terminal domain of TolB [27,43,51] and the central domain of TolR [51]. In the case of colicin A, the region of interaction with TolB, called the “TolB box”, is between residues 7 and 20 [43,51], the region of interaction with TolA consists of residues 52 and 97 [43], while residues 7–14 are involved in the interaction with TolR [51]. The TolB-box is conserved in several colicins that need TolB for their translocation step [27,43,44]. There is no sequence homology in the regions of interaction with TolA except for colicins A and K which suggests that these colicins interact with TolA in a similar way [52]. In the case of colicin N, the minimal TolA binding region is 27 residues [53]. Colicin E1 interacts with TolA differently than the other group A colicins [54]. The regions of interaction between TolB and TolR clearly overlap, suggesting that interaction between colicins and TolB and TolR is not simultaneous. This is not the case for the interactions with TolA and TolB since a ternary complex between colicin A, TolB and TolA has been characterized [43]. All these results have been summarized to propose a model for the translocation of colicin A (Fig. 2). The model takes into account the envelope localization of the Tol proteins and the structural information assuming that the structure of colicin A and Ia are similar. Colicin first binds to BtuB receptor via its central domain and unfolds (Fig. 2, step 1). The N-terminal translocation domain crosses the outer membrane near or in OmpF and the TolB box interacts with TolB which is close to the outer membrane due to its interaction with Pal (Fig. 2, step 2). As a consequence, TolB is no longer able to interact with TolA and Pal. This induces a tol phenotype and results in outer membrane fragility, allowing the colicin to further penetrate into the periplasm where it interacts with the C-terminus of TolA (Fig. 2, step 3). At this level a ternary complex between the colicin, TolA and TolB may occur. Colicin A then interacts with TolR near the inner membrane (Fig. 2, step 4). The mechanism of entry of the C-terminal catalytic domain is largely unknown. It is only known that colicin A spans the envelope and is still in contact with its outer membrane receptor when its translocation is completed [55]. Colicin A then forms a pore that depolarizes the inner membrane. Although this model can be generalized to colicins that use TolB for their
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Fig. 2. A proposed model for the translocation of colicin A across the cell envelope. For each step, proteins proposed to be involved in the translocation process are coloured in green. Colicin domains: T, translocation, R, receptor, C, catalytic. See text for comments of the model.
translocation, the way the colicins interact with Tol proteins may differ [4,54]. The energetics of the translocation is not fully understood. It has been reported that translocation of colicin A may be voltage-independent [56]. These experiments should probably be reconsidered in the light of the new results described above. It has been shown that the TolA C-terminal domain changes conformation when interacting with colicin A [57]. One can assume that the interaction of colicin A with the TolA C-terminal domain prevents this region of TolA from interacting with Pal and TolB, leading to the inactiva-
tion of the Tol system. Therefore, a possibility to reconcile these apparent inconsistencies would be that only the first step of colicin entry requires an energized Tol–Pal complex, a step that was probably by-passed in the experimental conditions described in Bourdineaud et al. [56]. Another interesting possibility is that presented in Journet et al. [51]. These authors propose that colicin translocation occurs by Brownian ratcheting. The forward movement of colicin would occur by simple diffusion, the Tol proteins would prevent it from moving backwards and would retain it by multiple interactions in the periplasm. This hypothesis can
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probably apply for the N-terminal domain of colicins and explain that the translocation may not require energy. However, it does not explain how the C-terminal region of colicins crosses the outer membrane. Translocation of large proteins such as colicins across bacterial envelopes is a complex mechanism. Although significant work has been done to understand this mechanism, many aspects are still unsolved. For instance, some experimental reports do not fully agree with the three functional domain organization of colicins. The central domain of colicins may not be the only region involved in receptor binding. In the case of colicin N, the C-terminal pore-forming region also binds trimeric porins [58]. A hybrid construct containing the translocation and receptor domains of the g3p protein from phage f1 and the catalytic site of colicin E3 still requires TolB for its translocation, suggesting that TolB may be also involved in the entry of the C-terminal domain of colicin E3 [59]. At least four important questions remain to be answered. (i) How does colicin cross the outer membrane after interaction with their receptor? (ii) What is the exact stoichiometry of the Tol proteins? A model has been proposed where four TolQ and two TolR proteins interact with one TolA molecule [40]. Pal and TolB may dimerize [36,37]. (iii) Is the proposed Brownian ratchet sufficient to achieve colicin translocation [51]? (iv) How do enzymatic colicins, which need to cross the inner membrane, reach their final target [60,61]? The Tol system constitutes a fascinating model to understand the mechanisms used by large molecules to cross the cell envelope. As in the case of the Ton system, the Tol system appears to use an ion potential from the inner membrane to induce a conformational change of TolA in the periplasm, leading to an interaction between inner and outer membrane proteins. This kind of signalization may contribute to the role of the Tol system in maintaining the outer membrane stability. In the case of the Ton system, an energy-dependent process involving the C-terminal domain of TonB and the N-terminal domain of outer membrane receptors, leads to the opening of closed channel, allowing the uptake of iron siderophore or cobalamin. In the case of the Tol system, it is reasonable to assume that the same kind of energy-dependent process involves the C-terminal domain of TolA and an interaction with Pal and TolB (although the energy-dependence of the interaction with TolB has not been demonstrated). The tol phenotype strongly suggests that the Tol–Pal proteins are involved in outer membrane stabilization, either by contributing to a structural network between the outer membrane and the peptidoglycan, or by contributing to the localization of some outer membrane components. These two possibilities are not mutually exclusive. Colicins use the Ton and Tol systems for their translocation into the bacterial cell. While the Ton system is clearly involved in bacterial iron uptake, the exact role of the Tol system remains to be discovered. The high degree of conservation of the tol–pal operon, and the fact that some of
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the tol–pal genes appear to be required for cell viability in organisms such as Pseudomonas aeruginosa [62], Haemophilus. ducreyi [63] or E. coli O157 [64], probably reflects an important role of the Tol system in cell envelope biogenesis. In addition, the involvement of the Tol proteins in the uptake of phage DNA is not necessarily deleterious for the bacteria, since it is required for the horizontal gene transfer of genes involved in virulence [12]. Finally, a major difference between the Ton and Tol system is that the later is likely to allow both the uptake and export of biomolecules.
Acknowledgements Work in our laboratory was supported by the Life Science Department of the CNRS, the University of Lyon and a joint BQR between the University and the INSA of Lyon. JFD has an MENRT fellowship.
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